Neuronal death as a consequence of stroke or neurodegenerative disorders occurs by both acute failure in function (i.e, necrosis) and inappropriate activation of a genetic death program (i.e., apoptosis). The latter process is addressed, with the focus being to understand the regulatory events that promote Bax-dependent cell death. The pro-apoptotic protein Bax plays a crucial role in apoptosis through facilitating cytochrome c release from mitochondria, the result of which is proteolytic destruction of the cell. This proposal tests the hypothesis that mitochondrial outer membrane Bax pores form only after two crucial events occur- induction of a cytoplasmic Bax transformation known as the 6A7 conformational change, and unmasking of a Bax mitochondrial binding site. The long term goal of this research is to understand the regulatory mechanisms that trigger neuronal apoptosis. Cerebellar granule neurons isolated from newborn rats will be used as a model to study Bax-dependent apoptosis. Depriving these neurons of serum growth factors and depolarizing extracellular K+ (i.e., incubation with 3.5 mM K+ without serum, referred to as K+/serum deprivation) initiates the death program. The first specific aim is to establish the mitochondrial and cytoplasmic distributions of Bax elicited by K+ and/or serum deprivation, to quantify the amount of Bax that binds, and to further determine if a conformational change associated with cytochrome c release occurs before or after translocation to the mitochondria. Increased steady- state free radicals/reactive oxygen species are commonly observed during neuronal apoptosis, but their role in apoptosis is unclear. The second specific aim is to determine to what extent mitochondrial inner membrane free radicals regulate the Bax transition, with particular attention being focused on the translocation step. Mitochondrial-targeted antioxidants will be used to test this possibility. Bax may associate with mitochondria through the mitochondrial outer membrane voltage-dependent anion channel, or VDAC, at specific junctional complexes known as contact sites. Hexokinase and creatine kinase are regulated components of these sites that may preclude or compete with Bax binding. If true, it is predicted that the mitochondrial content of one or both kinases decreases during apoptosis. The third specific aim is to investigate the cytoplasmic/mitochondrial distribution of hexokinase as a way to assess changes in the number of VDAC sites that may be available for Bax, and to additionally determine the protein composition of contact sites during apoptosis. Data obtained from these aims will improve our understanding of the regulatory events controlling Bax in the early stages of apoptosis. The experimental approaches to address these aims include quantitative immunofluorescence, immunoprecipitation, and Western blotting. [unreadable] PUBLIC HEALTH RELEVANCE: Inappropriate neuronal apoptosis contributes to the severity of brain damage resulting from stroke and neurodegenerative disorders such as Alzheimer's disease and Amyotrophic Lateral Sclerosis. The incidence of these conditions is high: approximately 4,000,000 stroke survivors must cope with the debilitating effects of neuronal death and approximately 4,500,000 people currently suffer from Alzheimer's disease, a number that has doubled since 1980. Data obtained from this proposal will contribute to understanding the mechanisms responsible for neuronal apoptosis, thus aiding drug development to limit the severity of these pathologies. [unreadable] [unreadable]